PROJECT SUMMARY/ABSTRACT: IWITH-IN As many as 75% of long-term pediatric liver allografts harbor chronic portal inflammation, interface hepatitis, and/or fibrosis (peri-portal and/or peri-venular), even in the face of normal liver tests. Substantial gaps in knowledge remain as to the natural history, risk factors, and mechanisms of subclinical, progressive, chronic allograft deterioration. Interventions to mitigate inexorable allograft damage must be informed by definitive data regarding sensitive and specific biomarkers of progressive allograft injury and/or detailed knowledge regarding injury mechanism(s). To attack the problem of structural deterioration of pediatric liver allografts, our proposed ancillary study will utilize a unique and invaluable set of longitudinal data and tissue (blood and biopsy) specimens from subjects identified as INeligible to proceed to immunosuppression withdrawal in the parent iWITH study. The data and specimens will be collected during the tenure of and made available by the leadership of the parent iWITH study. This proposal responds eloquently to the Funding Opportunity Announcement PAR-16-034 as it leverages unique and rich information available for the iWITH-IN cohort that are the focus of this ancillary study proposal. This proposal calls for a direct and detailed interrogation of the allograft microenvironment, to identify demographic, clinical, serological, and histological characteristics associated with allograft stability versus progression as determined by a central pathologist's assessment of sequential surveillance liver biopsies separated by 4-5 years. Pre-designed multiplex staining panels will be used to characterize parenchymal, non- parenchymal, and infiltrating cell populations. These phenotypic data will be complemented by and correlated with transcriptional profiling data indicative of the allograft's functional status as well as serological data indicative of the humoral immune response to the allograft. Finally, sophisticated statistical analytical platforms will be utilized to integrate clinical data with the histological, serological, immunohistochemical, and transcriptional data streams to generate cross-platform risk classifiers and/or elucidate mechanisms of progressive allograft damage. The innovation of this proposal is embodied in the longitudinal liver biopsies to delineate patterns of allograft deterioration and the cutting-edge cross-platform analyses of rich and varied datasets. These essential resources are absolutely without parallel. Moreover, the PI has recruited and led a team of clinical and translational investigators with stellar records of expertise and experience. The knowledge gained will yield the requisite insight to guide the rational design of intervention(s) to arrest, reverse, and/or prevent subclinical chronic allograft damage that threatens the health and longevity of pediatric liver transplant recipients.